PROJECT SUMMARY AND ABSTRACT The ability to use a virus as a carrier particle to deliver DNA vaccines directly to antigen presenting cells has not been previously explored. Our proposal seeks to develop a virus-DNA-virus complex vaccine that will be preferentially taken up by dendritic cells (DCs) and other important Antigen Presenting Cells (APC) in vivo, that will deliver intracellular nucleic acid, activate DCs by engaging toll-receptor- like (TLR) signaling pathways, and deliver a vaccine stimulus that should promote robust and long- term immune activation against Influenza A pathogens. Targeted antigen presenting cell (APC) uptake and activation needs to occur to foster an appropriate immune activation sequence to pathogen subunit vaccine antigens that are by themselves weakly immunogenic. We have exploited the stability, and APC uptake properties of Tobacco Mosaic Virus (TMV) to deliver DNA by macropinocytosis. Our preliminary data demonstrate that TMV virus is actively taken up DCs, and induce DC and other APC activation pathways. TMV has been engineered to deliver DNA encoding Ovalbumin, expressed from a CMV promoter (TMV-OvaD). Preparation of TMV-OvaD particles, is a simple two step process, and engineered to deliver intracellular DNA after uptake. Based on immunogenicity in mice, TMV-OvaD can induce significantly improved IgG and cytotoxic T lymphocyte (CTL) immune responses to Ova protein or SIINFEKL peptide, compared to CMV- Ova intramuscular injection. However, there are several crucial steps to optimize TMV-DNA antigen delivery, and boost immune response activation. CMV promoter-driven expression is limited to nuclear RNA transcription, and may limit antigen expression. First, we want to explore the use of a CMV promoter to drive expression of a small self-replicating RNA, which will promote and amplify cytoplasmic RNA transcription, boost antigen delivery, and provide a second TLR signal. Second, we will move from model antigen expression (Ova and GFP), to expressing influenza virus Hemagglutinin (HA) antigens relevant to pandemic and pre-pandemic influenza (H5N1 and H7N9 HAs). Third, we will test TMV-DNA formulations in Influenza pathogen challenge studies to determine protective efficacy, and initiate safety testing in mice. We believe that TMV-DNA vaccines will target APC uptake, provide several sources of TLR signaling and activation, and stimulate appropriate antigen presentation context that stimulates broadly neutralizing antibodies. Our goal is to actualize the promise of safe and effective rapid response influenza vaccines against potentially pandemic disease. Development of similar vaccines against other pathogens, where complex mixtures of antigens may be required for stimulating adequate immunity, may broaden the utility of this approach.